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DNA Sequencing

In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Thus far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as Pyrosequencing are gaining an increasing share of the sequencing market. More genome data is being produced by pyrosequencing than Sanger DNA sequencing these days. Pyrosequencing has enabled rapid genome sequencing. Bacterial genome can be sequenced in a single run with several X coverage with this technique. This technique was also used to sequence the genome of James Watson recently.

DNA sequencing
The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in 'pure' research into why and how organisms live, as well as in applied subjects. Because of the key nature of DNA to living things, knowledge of DNA sequence may come in useful in practically any biological research. For example, in medicine it can be used to identify, diagnose and potentially develop treatments for genetic diseases. Similarly, research into pathogens may lead to treatments for contagious diseases. Biotechnology is a burgeoning discipline, with the potential for many useful products and services.

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Thus far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as Pyrosequencing are gaining an increasing share of the sequencing market. More genome data is being produced by pyrosequencing than Sanger DNA sequencing these days. Pyrosequencing has enabled rapid genome sequencing. Bacterial genome can be sequenced in a single run with several X coverage with this technique. This technique was also used to sequence the genome of James Watson recently.

Sanger sequencing
In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, an enzyme that replicates DNA. Included with the primer and DNA polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.

An alternative to the labelling of the primer is to label the terminators instead, commonly called 'dye terminator sequencing'. The major advantage of this approach is the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to a template dependent difference in the incorporation of the large dye chain-terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability.

This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper. The major reason for this is that the primers do not have to be separately labelled (which can be a significant expense for a single-use custom primer), although this is less of a concern with frequently used 'universal' primers.

Pyrosequencing
Pyrosequencing, which was originally developed by Mostafa Ronaghi, has been commercialized by Biotage (for low throughput sequencing) and 454 Life Sciences (for high-throughput sequencing). The latter platform sequences roughly 100 megabases in a 7-hour run with a single machine. In the array-based method (commercialized by 454 Life Sciences), single-stranded DNA is annealed to beads and amplified via emPCR. These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary base pairs. Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow.

Endoplasmic reticulum

The endoplasmic reticulum or ER is an organelle found in all eukaryotic cells that is an interconnected network of tubules, vesicles and cisternae. These structures are responsible for several specialized functions: Protein translation, folding, and transport of proteins to be used in the cell membrane (e.g., transmembrane receptors and other integral membrane proteins), or to be secreted (exocytosed) from the cell (e.g., digestive enzymes); sequestration of calcium; and production and storage of glycogen, steroids, and other macromolecules.The endoplasmic reticulum is part of the endomembrane system. The basic structure and composition of the ER membrane is similar to the plasma membrane.

Structure The general structure of the endoplasmic reticulum is an extensive membrane network of cisternae (sac-like structures) held together by the cytoskeleton. The phospholipid membrane encloses a space, the cisternal space (or lumen), from the cytosol. The functions of the endoplasmic reticulum vary greatly depending on the exact type of endoplasmic reticulum and the type of cell in which it resides. The three varieties are called rough endoplasmic reticulum, smooth endoplasmic reticulum, and sarcoplasmic reticulum.

Rough endoplasmic reticulum
The surface of the rough endoplasmic reticulum is studded with protein-manufacturing ribosomes giving it a "rough" appearance (hence its name).But it should be noted that these ribosomes are not resident of the endoplasmic reticulum initially. The ribosomes only bind to the ER once it begins to synthesize a protein destined for sorting. The free ribosome begins producing the polypeptide until a cytosolic signal recognition particle recognizes the pre-piece of 5-15 hydrophobic AAs preceded by a positively charged (basic) amino acid. This makes it easy for the complex to loop the sequence through the hydrophobic membrane. The pre-piece is then cleaved off.

The membrane of the rough endoplasmic reticulum is continuous with the outer layer of the nuclear envelope. Although there is no continuous membrane between the rough ER and the Golgi apparatus, membrane bound vesicles shuttle proteins between these two compartments. COP II brings vesicles to the golgi and COP I brings the membrane back. The rough endoplasmic reticulum works in concert with the Golgi complex to target new proteins to their proper destinations.

The RER is key in producing
lysosomal enzymes with a Mannose-6-phosphate marker added in the cis-golgi network
Secreted proteins, either secreted constitutively with no tag, or regulated secretion involving clathrin and paired basic amino acids in the signal peptide.
integral membrane proteins that stay imbedded in the membrane as vesicles exit and bind to new membranes. Rab proteins are key in targeting the membrane, SNAP and SNARE proteins are key in the fusion event.
initial glycosylation as assembly continues. This is either N-linked or O-linked (O-linked may likely occur in the golgi).
N-linked glycosylation: if the protein is properly folded, glycosyltransferase recognizes the AA sequence NXS or NXT (with the S/T residue phosphorylated) and adds a 14 sugar backbone (2 N-acetylglucosamine, 9 branching mannose, and 3 glucose at the end) to the side chain nitrogen of Asn.

Smooth endoplasmic reticulum (SER)
The smooth endoplasmic reticulum has functions in several metabolic processes, including synthesis of lipids, metabolism of carbohydrates and calcium concentration, drug detoxification, and attachment of receptors on cell membrane proteins. It is connected to the nuclear envelope. Smooth endoplasmic reticulum is found in a variety of cell types (both animal and plant) and it serves different functions in each. The Smooth ER also contains the enzyme Glucose-6-phosphatase which converts Glucose-6-phosphate to Glucose, a step in gluconeogenesis. The Smooth ER consists of tubules and vesicles that branch forming a network. In some cells there are dilated areas like the sacs of rough endoplasmic reticulum. The network of smooth endoplasmic reticulum allows increased surface area for the action or storage of key enzymes and the products of these enzymes. The smooth endoplasmic reticulum is known for its storage of calcium ions in muscle cells.

Sarcoplasmic reticulum
The sarcoplasmic reticulum is a special type of smooth ER found in smooth and striated muscle. The only structural difference between this organelle and the smooth endoplasmic reticulum is the medley of protein they have, both bound to their membranes and drifting within the confines of their lumens. This fundamental difference is indicative of their functions: the smooth ER synthesizes molecules and the sarcoplasmic reticulum stores and pumps calcium ions.The sarcoplasmic reticulum contains large stores of calcium, which it sequesters and then releases when the cell is depolarized. This has the effect of triggering muscle contraction.

Functions
The endoplasmic reticulum serves many general functions, including the facilitation of protein folding and the transport of synthesized proteins in sacs called cisternae.
Correct folding of newly-made proteins is made possible by several endoplasmic reticulum chaperone proteins, including protein disulfide isomerase (PDI), ERp29, the Hsp70 family member Grp78, calnexin, calreticulin, and the peptidylpropyl isomerase family. Only properly-folded proteins are transported from the rough ER to the Golgi complex.

Transport of proteins
Secretory proteins, mostly glycoproteins, are moved across the endoplasmic reticulum membrane. Proteins that are transported by the endoplasmic reticulum and from there throughout the cell are marked with an address tag called a signal sequence. The N-terminus (one end) of a polypeptide chain (i.e., a protein) contains a few amino acids that work as an address tag, which are removed when the polypeptide reaches its destination. Proteins that are destined for places outside the endoplasmic reticulum are packed into transport vesicles and moved along the cytoskeleton toward their destination. The endoplasmic reticulum is also part of a protein sorting pathway. It is, in essence, the transportation system of the eukaryotic cell. The majority of endoplasmic reticulum resident proteins are retained in the endoplasmic reticulum through a retention motif. This motif is composed of four amino acids at the end of the protein sequence. The most common retention sequence is KDEL (lys-asp-glu-leu). However, variation on KDEL does occur and other sequences can also give rise to endoplasmic reticulum retention. It is not known if such variation can lead to sub-endoplasmic reticulum localizations. There are three KDEL receptors in mammalian cells, and they have a very high degree of sequence identity. The functional differences between these receptors remain to be established.

Other functions
Insertion of proteins into the endoplasmic reticulum membrane: Integral proteins must be inserted into the endoplasmic reticulum membrane after they are synthesized. Insertion into the endoplasmic reticulum membrane requires the correct topogenic sequences.
Glycosylation: Glycosylation involves the attachment of oligosaccharides.
Disulfide bond formation and rearrangement: Disulfide bonds stabilize the tertiary and quaternary structure of many proteins.
Drug Detoxification: The smooth ER is the site at which some drugs are detoxified.

Mitochondria and Chloroplasts (the power generators)

Mitochondria and Chloroplasts (the power generators) Mitochondria are self-replicating organelles that occur in various numbers, shapes, and sizes in the cytoplasm of all eukaryotic cells. As mitochondria contain their own genome that is separate and distinct from the nuclear genome of a cell, they play a critical role in generating energy in the eukaryotic cell, they give the cell energy by the process of respiration, adding oxygen to food (typicially pertaining to glucose and ATP) to release energy. Organelles that are modified chloroplasts are broadly called plastids, and are often involved in storage. Since they contain their own genome, they are thought to have once been separate organisms, which later formed a symbiotic relationship with the cells. Chloroplasts are the counter-part of the mitochondria. Instead of giving off CO2 and H2O Plants give off glucose, oxygen, 6 molecules of water (compared to 12 in respiration) this process is called photosynthesis.

Cell nucleus

The cell nucleus is the most conspicuous organelle found in a eukaryotic cell. It houses the cell's chromosomes, and is the place where almost all DNA replication and RNA synthesis occur. The nucleus is spherical in shape and separated from the cytoplasm by a double membrane called the nuclear envelope. The nuclear envelope isolates and protects a cell's DNA from various molecules that could accidentally damage its structure or interfere with its processing. During processing, DNA is transcribed, or copied into a special RNA, called mRNA. This mRNA is then transported out of the nucleus, where it is translated into a specific protein molecule. In prokaryotes, DNA processing takes place in the cytoplasm.

Eukaryotic cells

Eukaryotic cells are about 10 times the size of a typical prokaryote and can be as much as 1000 times greater in volume. The major difference between prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific metabolic activities take place. Most important among these is the presence of a cell nucleus, a membrane-delineated compartment that houses the eukaryotic cell's DNA. It is this nucleus that gives the eukaryote its name, which means "true nucleus". Other differences include:

The plasma membrane resembles that of prokaryotes in function, with minor differences in the setup. Cell walls may or may not be present.
The eukaryotic DNA is organized in one or more linear molecules, called chromosomes, which are associated with histone proteins. All chromosomal DNA is stored in the cell nucleus, separated from the cytoplasm by a membrane. Some eukaryotic organelles also contain some DNA.
Eukaryotes can move using cilia or flagella. The flagella are more complex than those of prokaryotes.

Prokaryotic cells

Prokaryotes are distinguished from eukaryotes on the basis of nuclear organization, specifically their lack of a nuclear membrane. Prokaryotes also lack most of the intracellular organelles and structures that are characteristic of eukaryotic cells (an important exception is the ribosome, which are present in both prokaryotic and eukaryotic cells). Most functions of organelles, such as mitochondria, chloroplasts, and the Golgi apparatus, are taken over by the prokaryotic plasma membrane. Prokaryotic cells have three architectural regions: appendages called flagella and pili — proteins attached to the cell surface; a cell envelope consisting of a capsule, a cell wall, and a plasma membrane; and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions. Other differences include:

• The plasma membrane (a phospholipid bilayer) separates the interior of the cell from its environment and serves as a filter and communications beacon.
  
• Most prokaryotes have a cell wall (some exceptions are Mycoplasma (a bacterium) and Thermoplasma (an archaeon)). It consists of peptidoglycan in bacteria, and acts as an additional barrier against exterior forces. It also prevents the cell from "exploding" (cytolysis) from osmotic pressure against a hypotonic environment. A cell wall is also present in some eukaryotes like plants (cellulose) and fungi, but has a different chemical composition.
  
• A prokaryotic chromosome is usually a circular molecule (an exception is that of the bacterium Borrelia burgdorferi, which causes Lyme disease). Even without a real nucleus, the DNA is condensed in a nucleoid. Prokaryotes can carry extrachromosomal DNA elements called plasmids, which are usually circular. Plasmids can carry additional functions, such as antibiotic resistance.

Subcellular components of Cell

All cells, whether prokaryotic or eukaryotic, have a membrane that envelops the cell, separates its interior from its environment, regulates what moves in and out (selectively permeable), and maintains the electric potential of the cell. Inside the membrane, a salty cytoplasm takes up most of the cell volume. All cells possess DNA, the hereditary material of genes, and RNA, containing the information necessary to build various proteins such as enzymes, the cell's primary machinery. There are also other kinds of biomolecules in cells. This article will list these primary components of the cell, then briefly describe their function.

Cell membrane: A cell's defining boundary
The cytoplasm of a cell is surrounded by a plasma membrane. The plasma membrane in plants and prokaryotes is usually covered by a cell wall. This membrane serves to separate and protect a cell from its surrounding environment and is made mostly from a double layer of lipids (hydrophobic fat-like molecules) and hydrophilic phosphorus molecules. Hence, the layer is called a phospholipid bilayer. It may also be called a fluid mosaic membrane. Embedded within this membrane is a variety of protein molecules that act as channels and pumps that move different molecules into and out of the cell. The membrane is said to be 'semi-permeable', in that it can either let a substance (molecule or ion) pass through freely, pass through to a limited extent or not pass through at all. Cell surface membranes also contain receptor proteins that allow cells to detect external signalling molecules such as hormones.

Cytoskeleton: A cell's scaffold
The cytoskeleton acts to organize and maintain the cell's shape; anchors organelles in place; helps during endocytosis, the uptake of external materials by a cell, and cytokinesis, the separation of daughter cells after cell division; and moves parts of the cell in processes of growth and mobility. The eukaryotic cytoskeleton is composed of microfilaments, intermediate filaments and microtubules. There is a great number of proteins associated with them, each controlling a cell's structure by directing, bundling, and aligning filaments. The prokaryotic cytoskeleton is less well-studied but is involved in the maintenance of cell shape, polarity and cytokinesis.

Genetic material
Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most organisms use DNA for their long-term information storage, but some viruses (e.g., retroviruses) have RNA as their genetic material. The biological information contained in an organism is encoded in its DNA or RNA sequence. RNA is also used for information transport (e.g., mRNA) and enzymatic functions (e.g., ribosomal RNA) in organisms that use DNA for the genetic code itself.
Prokaryotic genetic material is organized in a simple circular DNA molecule (the bacterial chromosome) in the nucleoid region of the cytoplasm. Eukaryotic genetic material is divided into different, linear molecules called chromosomes inside a discrete nucleus, usually with additional genetic material in some organelles like mitochondria and chloroplasts (see endosymbiotic theory).
A human cell has genetic material in the nucleus (the nuclear genome) and in the mitochondria (the mitochondrial genome). In humans the nuclear genome is divided into 46 linear DNA molecules called chromosomes. The mitochondrial genome is a circular DNA molecule separate from the nuclear DNA. Although the mitochondrial genome is very small, it codes for some important proteins.
Foreign genetic material (most commonly DNA) can also be artificially introduced into the cell by a process called transfection. This can be transient, if the DNA is not inserted into the cell's genome, or stable, if it is.

Organelles
The human body contains many different organs, such as the heart, lung, and kidney, with each organ performing a different function. Cells also have a set of "little organs," called organelles, that are adapted and/or specialized for carrying out one or more vital functions. Membrane-bound organelles are found only in eukaryotes.

Credit by Wikipedia

Intro about Cell

The cell is the structural and functional unit of all known living organisms. It is the smallest unit of an organism that is classified as living, and is sometimes called the building block of life.Some organisms, such as bacteria, are unicellular (consist of a single cell). Other organisms, such as humans, are multicellular. (Humans have an estimated 100 trillion or 1014 cells; a typical cell size is 10 µm; a typical cell mass is 1 nanogram.) The largest known cell is an ostrich egg. In 1837 before the final cell theory was developed, a Czech Jan Evangelista Purkyňe observed small "granules" while looking at the plant tissue through a microscope. The cell theory, first developed in 1839 by Matthias Jakob Schleiden and Theodor Schwann, states that all organisms are composed of one or more cells. All cells come from preexisting cells. Vital functions of an organism occur within cells, and all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells.
The word cell comes from the Latin cellula, meaning, a small room. The descriptive name for the smallest living biologial structure was chosen by Robert Hooke in a book he published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in.

RNAi

RNA interference (also called "RNA-mediated interference", abbreviated RNAi) is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. Conserved in most eukaryotic organisms, the RNAi pathway is thought to have evolved as a form of innate immunity against viruses and also plays a major role in regulating development and genome maintenance.
The RNAi pathway is initiated by the enzyme dicer, which cleaves double-stranded RNA (dsRNA) to short double-stranded fragments of 20–25 base pairs. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and base-pairs with complementary sequences. The most well-studied outcome of this recognition event is a form of post-transcriptional gene silencing. This occurs when the guide strand base pairs with a messenger RNA (mRNA) molecule and induces degradation of the mRNA by argonaute, the catalytic component of the RISC complex. The short RNA fragments are known as small interfering RNA (siRNA) when they derive from exogenous sources and microRNA (miRNA) when they are produced from RNA-coding genes in the cell's own genome. The RNAi pathway has been particularly well-studied in certain model organisms such as the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the flowering plant Arabidopsis thaliana.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms; synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.
Historically, RNA interference was known by other names, including post transcriptional gene silencing, transgene silencing, and quelling. Only after these apparently-unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. RNAi has also been confused with antisense suppression of gene expression, which does not act catalytically to degrade mRNA but instead involves single-stranded RNA fragments physically binding to mRNA and blocking translation. In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm C. elegans,which they published in 1998.

Cellular mechanism

RNAi is an RNA-dependent gene silencing process that is mediated by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in the cytoplasm, where they interact with the catalytic RISC component argonaute. When the dsRNA is exogenous, coming from infection by a virus with an RNA genome or laboratory manipulations, the RNA is imported directly into the cytoplasm and cleaved to short fragments by the enzyme dicer. The initiating dsRNA can also be endogenous, as in pre-microRNAs expressed from RNA-coding genes in the genome. The primary transcripts from such genes are first processed to the characteristic stem-loop structure of pre-miRNA in the nucleus, then exported to the cytoplasm to be cleaved by dicer. Thus the two pathways for exogenous and endogenous dsRNA converge at the RISC complex, which mediates gene silencing effects.

dsRNA cleavage

Exogenous dsRNA initiates RNAi by activating the ribonuclease protein dicer,which binds and cleaves double-stranded RNAs (dsRNA)s to produce double-stranded fragments of 20–25 base pairs with a few unpaired overhang bases on each end.Bioinformatics studies on the genomes of multiple organisms suggest this length maximizes target-gene specificity and minimizes non-specific effects.These short double-stranded fragments are called small interfering RNAs (siRNAs). These siRNAs are then separated into single strands and integrated into an active RISC complex. After integration into the RISC, siRNAs base-pair to their target mRNA and induce cleavage of the mRNA, thereby preventing it from being used as a translation template.
Exogenous dsRNA is detected and bound by an effector protein known as RDE-4 in C. elegans and R2D2 in Drosophila that stimulates dicer activity.This protein only binds long dsRNAs, but the mechanism producing this length specificity is unknown.These RNA-binding proteins then facilitate transfer of cleaved siRNAs to the RISC complex.
This initiation pathway may be amplified by the cell through the synthesis of a population of 'secondary' siRNAs using the dicer-produced initiating or 'primary' siRNAs as templates.These siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).

microRNA
MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that help regulate gene expression, particularly during development. The phenomenon of RNA interference, broadly defined, includes the endogenously induced gene silencing effects of miRNAs as well as silencing triggered by foreign dsRNA. Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA, but miRNAs must first undergo extensive post-transcriptional modification. An miRNA is expressed from a much longer RNA-coding gene as a primary transcript known as a pri-miRNA, which is processed in the cell nucleus to a 70-nucleotide stem-loop structure called a pre-miRNA by the microprocessor complex. This complex consists of an RNase III enzyme called Drosha and a dsRNA-binding protein Pasha. The dsRNA portion of this pre-miRNA is bound and cleaved by dicer to produce the mature miRNA molecule that can be integrated into the RISC complex; thus, miRNA and siRNA share the same cellular machinery downstream of their initial processing.
The siRNAs derived from long dsRNA precursors differ from miRNAs in that miRNAs, especially those in animals, typically have incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only in a single, specific target.In Drosophila and C. elegans, miRNA and siRNA are processed by distinct argonaute proteins and dicer enzymes.

RISC activation and catalysis
The catalytically-active components of the RISC complex are endonucleases called argonaute proteins, which cleave the target mRNA strand complementary to their bound siRNA. As the fragments produced by dicer are double-stranded, they could each in theory produce a functional siRNA. However, only one of the two strands, which is known as the guide strand, binds the argonaute protein and directs gene silencing. The other anti-guide strand or passenger strand is degraded during RISC activation.Although it was first believed that an ATP-dependent helicase separated these two strands,the process is actually ATP-independent and performed directly by the protein components of RISC.The strand selected as the guide tends to be that with a more stable 5' end, but strand selection is unaffected by the direction in which dicer cleaves the dsRNA before RISC incorporation.Instead, the R2D2 protein may serve as the differentiating factor by binding the less-stable 5' end of the passenger strand.The structural basis for binding of RNA to the argonaute protein was examined by X-ray crystallography of the binding domain of an RNA-bound argonaute protein. Here, the phosphorylated 5' end of the RNA strand enters a conserved basic surface pocket and makes contacts through a divalent cation such as magnesium and by aromatic stacking between the 5' nucleotide in the siRNA and a conserved tyrosine residue. This site is thought to form a nucleation site for the binding of the siRNA to its mRNA target.
It is not understood how the activated RISC complex locates complementary mRNAs within the cell. Although the cleavage process has been proposed to be linked to translation, translation of the mRNA target is not essential for RNAi-mediated degradation.Indeed, RNAi may be more effective against mRNA targets that are not translated.Argonaute proteins, the catalytic components of RISC, are localized to specific regions in the cytoplasm called P-bodies (also cytoplasmic bodies or GW bodies), which are regions with high rates of mRNA decay;miRNA activity is also clustered in P-bodies.Disruption of P bodies in cells decreases the efficiency of RNA interference, suggesting that they are the site of a critical step in the RNAi process.

Variation among organisms
Organisms vary in their ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNA interference can be both systemic and heritable in plants and C. elegans, although not in Drosophila or mammals. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata.A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNAs; in plants, miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and induce translational repression.This translational effect may be produced by inhibiting the interactions of translation initiation factors with the messenger RNA's polyadenine tail.Some eukaryotic protozoa such as Leishmania major and Trypanosoma cruzi lack the RNAi pathway entirely.Most or all of the components are also missing in some fungi, most notably the model organism Saccharomyces cerevisiae.Certain ascomycetes and basidiomycetes are also missing RNA interference pathways; this observation indicates that proteins required for RNA silencing have been lost independently from many fungal lineages, possibly due to the evolution of a novel pathway with similar function, or to the lack of selective advantage in certain niches.

Human Chromosome

Human Chromosome: Human cells have 23 pairs of large linear nuclear chromosomes, giving a total of 46 per cell. In addition to these, human cells have many hundreds of copies of the mitochondrial genome. Sequencing of the human genome has provided a great deal of information about each of the chromosomes. Below is a table compiling statistics for the chromosomes, based on the Sanger Institute's human genome information in the Vertebrate Genome Annotation (VEGA) database. Number of genes is an estimate as it is in part based on gene predictions. Total chromosome length is an estimate as well, based on the estimated size of unsequenced heterochromatin regions.

Chromosomes in prokaryotes

Chromosomes in prokaryotes: Prokaryotes (eg. Bacteria) typically have a single circular chromosome, but many variations do exist. Bacterial DNA also exists as plasmids, essentially miniature chromosomes, which are small circular pieces of DNA that are readily transmitted between bacteria. The distinction between plasmids and chromosomes is poorly defined, though size and necessity are generally taken into account.
Structure in sequences: Prokaryotes chromosomes have less sequence based structure than eukaryotes. They do, however, typically have a single point, the origin of replication, from which replication starts.
The genes in prokaryotes are often organised in operons, and do not contain introns, unlike eukaryotes.
Location in the cell: Bacterial chromosomes tend to be tethered to the plasma membrane of the bacteria. In molecular biology application, this allows for its isolation from plasmid DNA by centrifugation of lysed bacteria and pelleting of the membranes (and the attached DNA).
DNA packaging: Prokaryotes do not possess histones or nuclei, and so do not possess chromatin like eukaryotes. There is, however, thought to be some structural organisation to help condense the large molecule into the small prokaryotic cell.
Prokaryotic chromosomes and plasmids are, like eukaryotic DNA, generally supercoiled. The DNA must first be released into its relaxed state for access for transcription, regulation, and replication.

Chromosomes in eukaryotes

Chromosomes in eukaryotes: Eukaryotes (cells with nuclei such as plants, yeast, and animals) possess multiple large linear chromosomes contained in the cell's nucleus. Each chromosome has one centromere, with one or two arms projecting from the centromere, although under most circumstances these arms are not visible as such. In addition most eukaryotes have a small circular mitochondrial genome, and some eukaryotes may have additional small circular or linear cytoplasmic chromosomes.
In the nuclear chromosomes of eukaryotes, the uncondensed DNA exists in a semi-ordered structure, where it is wrapped around histones (structural proteins), forming a composite material called chromatin.
Chromatin: the complex of DNA and protein found in the eukaryotic nucleus which packages chromosomes. The structure of chromatin varies significantly between different stages of the cell cycle, according to the requirements of the DNA.
Interphase chromatin: During interphase (the period of the cell cycle where the cell is not dividing) two types of chromatin can be distinguished:
· Euchromatin, which consists of DNA that is active, e.g., expressed as protein.
· Heterochromatin, which consists of mostly inactive DNA. It seems to serve structural purposes during the chromosomal stages. Heterochromatin can be further distinguished into two types:
o Constitutive heterochromatin, which is never expressed. It is located around the centromere and usually contains repetitive sequences.
o Facultative heterochromatin, which is sometimes expressed.
Individual chromosomes cannot be distinguished at this stage - they appear in the nucleus as a homogeneous tangled mix of DNA and protein.

Metaphase chromatin and division: In the early stages of mitosis or meiosis (cell division), the chromatin strands become more and more condensed. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. This compact form makes the individual chromosomes visible, and they form the classic four arm structure, a pair of sister chromatids attach to each other at the centromere. The shorter arms are called p arms (from the French petit, small) and the longer arms are called q arms (q follows p in the Latin alphabet). This is the only natural context in which individual chromosomes are visible with an optical microscope.
During divisions long microtubules attach to the centromere and the two opposite ends of the cell. The microtubules then pull the chromatids apart, so that each daughter cell inherits one set of chromatids. Once the cells have divided, the chromatids are uncoiled and can function again as chromatin. In spite of their appearance, chromosomes are structurally highly condensed which enables these giant DNA structures to be contained within a cell nucleus.
The self assembled microtubules form the spindle, which attaches to chromosomes at specialized structures called kinetochores, one of which is present on each sister chromatid. A special DNA base sequence in the region of the kinetochores provides, along with special proteins, longer-lasting attachment in this region.

Intro about Chromosome

Chromosome is a single large macromolecule of DNA, and constitutes a physically organized form of DNA in a cell. It is a very long, continuous piece of DNA (a single DNA molecule), which contains many genes, regulatory elements and other intervening nucleotide sequences. A broader definition of "chromosome" also includes the DNA-bound proteins which serve to package and manage the DNA. The word chromosome comes from the Greek chroma is color and soma is body due to its capacity to be stained very strongly with vital and supravital dyes.
Chromosomes vary extensively between different organisms. The DNA molecule may be circular or linear, and can contain anything from tens of kilobase pairs to hundreds of megabase pairs. Typically eukaryotic cells (cells with nuclei) have large linear chromosomes and prokaryotic cells (cells without nuclei) smaller circular chromosomes, although there are many exceptions to this rule. Furthermore, cells may contain more than one type of chromosome; for example mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosome in addition to the nuclear chromosomes.
In eukaryotes nuclear chromosomes are packaged by proteins (particularly histones) into chromatin to fit the massive molecules into the nucleus. The structure of chromatin varies through the cell cycle, and is responsible for the compaction of DNA into the classic four-arm structure during mitosis and meiosis. Prokaryotes do not form chromatin, because the cells lack proteins required and the circular configuration of the molecule prevents this.
"Chromosome" is a rather loosely defined term. In prokaryotes, a small circular DNA molecule may be called either a plasmid or a small chromosome. In viruses, mitochondria, and chloroplasts their DNA molecules are commonly referred to as chromosomes, despite being naked molecules, as they constitute the complete genome of the organism or organelle.
History
Chromosomes were first observed in plant cells by a Swiss botanist named Karl Wilhelm von Nägeli in 1842, and independently in Ascaris worms by Belgian scientist Edouard Van Beneden (1846-1910). The use of basophilic aniline dyes was a fundamentally new technique for effectively staining the chromatin material in the nucleus. Their behavior in animal (salamander) cells was later described in detail by German cytologist and professor of anatomy Walther Flemming, the discoverer of mitosis, in 1882. The name was invented later by another German anatomist, Heinrich von Waldeyer.

RNA Splicing

In genetics, splicing is a modification of genetic information after transcription, in which introns are removed and exons are joined. Since in prokaryotic genomes introns do not exist, splicing naturally only occurs in eukaryotes. The splicing prepares the precursor messenger RNA (pre-mRNA) to produce the mature messenger RNA (mRNA), which then undergoes translation as part of the protein synthesis to produce proteins. Splicing includes a series of biochemical reactions, which are catalyzed by the spliceosome, a complex of small nuclear ribonucleo-proteins (snRNPs).
Splicing pathways: Several methods of RNA splicing occur in nature. The type of splicing depends on the structure of the spliced intron and the catalysts required for splicing to occur. Regardless of which pathway is used, the excised introns are discarded.
Spliceosomal introns
Spliceosomal introns often reside in eukaryotic protein-coding genes. Within the intron, a 3' splice site, 5' splice site, and branch site are required for splicing. Splicing is catalyzed by the spliceosome which is a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs, pronounced 'snurps' ). The RNA components of snRNPs interact with the intron and may be involved in catalysis. Two types of spliceosomes have been identified (the major and minor) which contain different snRNPs.
·Major
The major spliceosome splices introns containing GU at the 5' splice site and AG at the 3' splice site. It is composed of the U1, U2, U4, U5, and U6 snRNPs.
·E Complex-U1 binds to the GU sequence at the 5' splice site, along with accessory proteins/enzymes ASF/SF2, U2AF (binds at the Py-AG site), SF1/BBP (BBP=Branch Binding Protein);
·A Complex-U2 binds to the branch site, and ATP is hydrolyzed;
·B1 Complex-U5/U4/U6 trimer binds, and the U5 binds exons at the 5' site, with U6 binding to U2;
·B2 Complex-U1 is released, U5 shifts from exon to intron and the U6 binds at the 5' splice site;
·C1 Complex-U4 is released, U6/U2 catalyzes transesterification, U5 binds exon at 3' splice site, and the 5' site is cleaved, resulting in the formation of the lariat;
·C2 Complex-U2/U5/U6 remain bound to the lariat, and the 3' site is cleaved and exons are ligated using ATP hydrolysis. The spliced RNA is released and the lariat debranches.
This type of splicing is termed canonical splicing or termed the lariat pathway, which accounts for more than 99% of splicing. By contrast, when the intronic flanking sequences do not follow the GU-AG rule, noncanonical splicing is said to occur (see "minor spliceosome" below).
·Minor
The minor spliceosome is very similar to the major spliceosome, however it splices rare introns with different splice site sequences. Here, the 3' and 5' splice sites are AU and AC, respectively. While the minor and major spliceosomes contain the same U5 snRNP, the minor spliceosome has different, but functionally analogous snRNPs for U1, U2, U4, and U6, which are respectively called U11, U12, U4atac, and U6atac.
·Trans-splicing
Trans-splicing is a form of splicing that joins two exons that are not within the same RNA transcript.
Self-splicing: Self-splicing occurs for rare introns that form a ribozyme, performing the functions of the spliceosome by RNA alone. There are two kinds of self-splicing introns, Group I, and II. Group I and II introns perform splicing similar to the spliceosome without requiring any protein. This similarity suggests that Group I and II introns may be evolutionarily related to the spliceosome. Self-splicing may also be very ancient, and may have existed in an RNA world that was present before protein. Although the two splicing mechanisms described below do not require any proteins to occur, 5 additional RNA molecules and over 50 proteins are used and hydrolyzes many ATP molecules. The splicing mechanisms use ATP in order to accurately splice mRNA's. If the cell were to not use any ATP's, the process would be highly inaccurate and many mistakes would occur. Two transesterfications characterize the mechanism in which group I introns are sliced: 1) 3'OH of a free guanine nucleoside (or one located in the intron) or a nucleotide cofactor (GMP, GDP, GTP) attacks phosphate at the 5' splice site. 2) 3'OH of the 5'OH becomes a nucleophile and the second transesterfication results in the joining of the two exons. The mechanism in which group II introns are spliced (two transesterfication reaction like group I introns) is as follows: 1)The 2'OH of a specific adenosine in the intron attacks the 5' splice site, thereby forming the lariat 2) The 3'OH of the 5' exon triggers the second transesterfication at the 3' splice site thereby joining the exons together.
tRNA splicing: tRNA (also tRNA-like) splicing is another rare form of splicing that usually occurs in tRNA. The splicing reaction involves a different biochemistry than the spliceomsomal and self-splicing pathways. Ribonucleases cleave the RNA and ligases join the exons together. This form of splicing does also not require any RNA components for catalysis.
Evolution: Splicing occurs in all the kingdoms or domains of life, however, the extent and types of splicing can be very different between the major divisions. Eukaryotes splice many protein-coding messenger RNAs and some non-coding RNAs. Prokaryotes, on the other hand, splice rarely, but mostly non-coding RNAs. Another important difference between these two groups of organisms is that prokaryotes completely lack the spliceosomal pathway.
Because spliceosomal introns are not conserved in all species, there is debate concerning when spliceosomal splicing evolved. Two models have been proposed: the intron late and intron early models (see intron evolution).
Biochemical mechanism: Diagram illustrating the two-step biochemistry of splicing
Spliceosomal splicing and self-splicing involves a two-step biochemical process. Both steps involve transesterification reactions that occur between RNA nucleotides. tRNA splicing, however, is an exception and does not occur by transesterification.
Spliceosomal and self-splicing transesterification reactions occur via two sequential transesterification reactions. First, the 2'OH of a specific branch-point nucleotide within the intron that is defined during spliceosome assembly performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site forming the lariat intermediate. Second, the 3'OH of the released 5' exon then performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat.
Experimental manipulation of splicing: Splicing events can be experimentally altered by binding steric-blocking antisense oligos such as Morpholinos or Peptide nucleic acids to snRNP binding sites, to the branchpoint nucleotide that closes the lariat, or to splice-regulatory element binding sites.
Splicing errors: Mutations in the introns or exons can prevent splicing and thus may prevent protein biosynthesis.
Common errors:
·Mutation of a splice site resulting in loss of function of that site. Results in exposure of a premature stop codon, loss of an exon, or inclusion of an intron.
·Mutation of a splice site reducing specificity. May result in variation in the splice location, causing insertion or deletion of amino acids, or most likely, a loss of the reading frame.
·Transposition of a splice site, resulting in inclusion or exclusion of more DNA than expected. Results in longer or shorter exons.

rRNA

Ribosomal RNA (rRNA): is a component of the ribosomes, the protein synthetic factories in the cell. Eukaryotic ribosomes contain four different rRNA molecules: 18 s, 5.8 s, 28 s, and 5 s rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere. rRNA molecules are extremely abundant. They make up at least 80% of the RNA molecules found in a typical eukaryotic cell.
Synthesis of the three nucleolar rRNA molecules is unusual because they are made on one primary transcript that is chopped up into three mature rRNA molecules. These rRNA molecules and the 5 s rRNA combine with the ribosomal proteins in the nucleolus to form pre 40 s and pre 60 s ribosomal subunits. These pre-subunits are exported to the nucleus where they mature and assume their role in protein synthesis.
The rRNA molecules have several roles in protein synthesis. First, the 28 s rRNA has a catalytic role, it forms part of the peptidyl transferrase activity of the 60 s subunit. Second, 18s rRNA has a recognition role, involved in correct positioning of the mRNA and the peptidyl tRNA. Finally, the rRNA molecules have a structural role. They fold into three-dimensional shapes that form the scaffold on which the ribosomal proteins assemble. The model on the left shows a the three dimensional structure that the 5 s rRNA from the African frog, Xenopus laevis is thought to adopt.
The ribosome is composed of two subunits, named for how rapidly they sediment when subject to centrifugation. tRNA is sandwiched between the small and large subunits and the ribosome catalyzes the formation of a peptide bond between the 2 amino acids that are contained in the tRNA.
The ribosome also has 3 binding sites called A, P, and E.
· The A site in the ribosome binds to an aminoacyl-tRNA (a tRNA bound to an amino acid).
· The NH2 group of the aminoacyl-tRNA which contains the new amino acid, attacks the carboxyl group of peptidyl-tRNA (contained within the P site) which contains the last amino acid of the growing chain called peptidyl transferase reaction.
· The tRNA that was holding on the the last amino acid is moved to the E site, and what used to be the aminoacyl-tRNA is now the peptidyl-tRNA.
A single mRNA can be translated simultaneously by multiple ribosomes.

Prokaryotes: In Prokaryotes a small 30S ribosomal subunit contains the 16S rRNA.
The large 50S ribosomal subunit contains two rRNA species (the 5S and 23S rRNAs).
Bacterial 16S, 23S, and 5S rRNA genes are typically organized as a co-transcribed operon.
There may be one or more copies of the operon dispersed in the genome (for example, Escherichia coli has seven).
Archaea contains either a single rDNA operon or multiple copies of the operon.
Eukaryotes: Small subunit ribosomal RNA, 5' domain taken from the Rfam database. This example is RF00177
In contrast, Eukaryotes generally have many copies of the rRNA genes organized in tandem repeats; in humans approximately 300–400 rDNA repeats are present in five clusters (on chromosomes 13, 14, 15, 21 and 22).
The 18S rRNA in most eukaryotes is in the small ribosomal subunit, and the large subunit contains three rRNA species (the 5S, 5.8S and 28S rRNAs).
The tertiary structure of the small subunit ribosomal RNA (SSU rRNA) has been resolved by xray crystallography. The secondary structure of SSU rRNA contains 4 distinct domains -- the 5', central, 3' major and 3' minor domains. A model of the secondary structure for the 5' domain (500-800 nucleotides) is shown.

DNA Replication

The Biochemical Reactions: DNA replication begins with the "unzipping" of the parent molecule as the hydrogen bonds between the base pairs are broken.
Once exposed, the sequence of bases on each of the separated strands serves as a template to guide the insertion of a complementary set of bases on the strand being synthesized.
The new strands are assembled from deoxynucleoside triphosphates.
Each incoming nucleotide is covalently linked to the "free" 3' carbon atom on the pentose (figure) as
the second and third phosphates are removed together as a molecule of pyrophosphate (PPi).
The nucleotides are assembled in the order that complements the order of bases on the strand serving as the template.
Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so on.
When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule.
The Enzymes: A portion of the double helix is unwound by a helicase.
A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix. In eukaryotes, this molecule is called DNA polymerase delta (δ).
Because DNA synthesis can only occur 5' to 3', a molecule of a second type of DNA polymerase (epsilon, ε, in eukaryotes) binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase I then stitches these together into the lagging strand.
Speed of Replication
Bacteria: The single molecule of DNA that is the E. coli genome contains 4.7 x 106 nucleotide pairs. DNA replication begins at a single, fixed location in this molecule, the replication origin, proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. And thanks to the precision of the process (which includes a "proof-reading" function), the job is done with only about one incorrect nucleotide for every 109 nucleotides inserted. In other words, more often than not, the E. coli genome (4.7 x 106) is copied without error!
Eukaryotes: The average human chromosome contains 150 x 106 nucleotide pairs which are copied at about 50 base pairs per second. The process would take a month (rather than the hour it actually does) but for the fact that there are many places on the eukaryotic chromosome where replication can begin. Replication begins at some replication origins earlier in S phase than at others, but the process is completed for all by the end of S phase. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally forming two new molecules.
Control of Replication: With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?
An observation: When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.
Two control mechanisms have been identified — one positive and one negative. This redundancy probably reflects the crucial importance of precise replication to the integrity of the genome.
Licensing: positive control of replication
In order to be replicated, each origin of replication must be bound by:
an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process.
Accessory proteins called licensing factors. These accumulate in the nucleus during G1 of the cell cycle. They include:
Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with
MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated.
Once replication begins in S phase,
Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and destruction in proteasomes).
The MCM proteins leave in front of the advancing replication fork.
Geminin: negative control of replication
G2 nuclei also contain at least one protein — called geminin — that prevents assembly of MCM proteins on freshly-synthesized DNA (probably by blocking the actions of Cdt1).
As the cell completes mitosis, geminin is degraded so the DNA of the two daughter cells will be able to respond to licensing factors and be able to replicate their DNA at the next S phase.

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Central Dogma

The central dogma of molecular biology was first enunciated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970
The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.
In other words, 'once information gets into protein, it can't flow back to nucleic acid.'
The dogma is a framework for understanding the transfer of sequence information between sequential information-carrying biopolymers, in the most common or general case, in living organisms. There are 3 major classes of such biopolymers: DNA and RNA (both nucleic acids), and protein. There are 3×3 = 9 conceivable direct transfers of information that can occur between these. The dogma classes these into 3 groups of 3: 3 general transfers (believed to occur normally in most cells), 3 special transfers (known to occur, but only under abnormal conditions), and 3 unknown transfers (believed to never occur). The general transfers describe the normal flow of biological information: DNA can be copied to DNA (DNA replication), DNA information can be copied into mRNA, (transcription), and proteins can be synthesized using the information in mRNA as a template (translation).